The redox state of cysteine residues can have a profound effect on protein structure and function [1-4]. Consequently, reagents that reduce disulfide bonds to thiols can be crucial to progress in chemical biology [1, 5-6]. The reduction of disulfide bonds within biomolecules is preferably accomplished under mild conditions: in water, at neutral pH, and at room temperature [1, 7-10]. Thiols can accomplish these goals and do so (unlike phosphines) in a reversible manner. The mechanism for reduction involves thiol-disulfide interchange initiated by the attack of a thiolate [11-18]. The use of monothiols such as L-glutathione or β mercaptoethanol (βME) can lead to the trapping of the resulting intermediate as a mixed disulfide. Cleland reported that dithiothreitol (DTT or Cleland's reagent, Table 1), a racemic dithiol, readily completes the reduction reaction by forming a stable six-membered ring [7]. The potency of DTT is evident from the low reduction potential (=−0.327 V) of its oxidized form [19]. As a result, DTT has achieved widespread use for the quantitative reduction of disulfide bonds in proteins and other biomolecules.
DTT has, however, a serious limitation. Because thiolates, but not thiols, are nucleophilic in aqueous solution [20], the observed rate of disulfide reduction is dependent on the thiol pKa of the reducing agent. With thiol pKa values of 9.2 and 10.1, less than 1% of DTT resides are in the reactive thiolate form at neutral pH.8. As a result, several attempts have been made to create water-soluble reducing agents that exhibit depressed thiol pKa values [9, 21-22].
Recently, dithiobutylamine (DTBA, Table 1), a dithiol reducing agent derived from L-aspartic acid was described as a potent disulfide-reducing agent [23]. Moreover, the amino group of DTBA confers depressed thiol pKa values of 8.2 and 9.3 and facile functionalization [23-24]. That amino group, however, appeared to deter the ability of the molecule to reduce certain disulfide bonds due to unfavorable Coulombic interactions [23].